DOI 10.1007/s00262-010-0916-z
O R I G I N A L A R T I C L E
MAGE-A3 and MAGE-A4 speci
Wc CD4
+
T cells in head and neck
cancer patients: detection of naturally acquired responses
and identi
Wcation of new epitopes
Valérie Cesson · Jean-Paul Rivals · Anette Escher · Elsa Piotet · Kris Thielemans · Vilmos Posevitz · Danijel Dojcinovic · Philippe Monnier · Daniel Speiser · Luc Bron · Pedro Romero
Received: 23 July 2010 / Accepted: 31 August 2010 / Published online: 21 September 2010 © Springer-Verlag 2010
Abstract Frequent expression of cancer testis antigens (CTA) has been consistently observed in head and neck squamous cell carcinomas (HNSCC). For instance, in 52 HNSCC patients, MAGE-A3 and -A4 CTA were expressed in over 75% of tumors, regardless of the sites of primary tumors such as oral cavity or hypopharynx. Yet, T-cell responses against these CTA in tumor-bearing patients have not been investigated in detail. In this study, we assessed the naturally acquired T-cell response against MAGE-A3 and -A4 in nonvaccinated HNSCC patients. Autologous antigen-presenting cells pulsed with overlap-ping peptide pools were used to detect and isolate MAGE-A3 and MAGE-A4 speciWc CD4+ T cells from healthy
donors and seven head and neck cancer patients. CD4+
T-cell clones were characterized by cytokine secretion. We could detect and isolate MAGE-A3 and MAGE-A4 speciWc CD4+ T cells from 7/7 cancer patients analyzed. Moreover, we identiWed six previously described and three new epitopes for MAGE-A3. Among them, the MAGE-A3111–125
and MAGE-A3161–175 epitopes were shown to be naturally processed and presented by DC in association with HLA-DP and DR, respectively. All of the detected MAGE-A4 responses were speciWc for new helper epitopes. These data suggest that naturally acquired CD4+ T-cell responses against CT antigens often occur in vivo in HNSCC cancer patients and provide a rationale for the development of active immunotherapeutic approaches in this type of tumor. Keywords HNSCC · MAGE-A3 · MAGE-A4 ·
CD4+ T cells Abbreviations
pMHC Peptide-MHC
HNSCC Head and neck squamous cell carcinomas HD Healthy donor
Introduction
Squamous cell carcinoma of the head and neck (HNSCC) represents the Wfth most common cancer worldwide and is a signiWcant cause of cancer morbidity and mortality. A majority of patients with HNSCC present with advanced disease at the time of Wrst evaluation. While early stages of HNSCC are generally curable with surgery and chemoradi-ation, the overall survival for patients with HNSCC is poor and has not appreciably improved in the past three decades. Therefore, novel therapeutic approaches are urgently needed and recent research has focused on the development of immunotherapy to complement conventional treatments. Frequent expression of tumor-associated antigens (TAA) has been repeatedly observed in head and neck carcinomas. These observations have led to the design of targeted cancer vaccine trials [1]. TAA are derived from a broad spectrum V. Cesson · V. Posevitz · D. Speiser · P. Romero (&)
Ludwig Institute for Cancer Research Ltd., Lausanne Branch, University Hospital (CHUV), Hôpital Orthopédique Niv. 5, aile est, Av. Pierre Decker 4, 1011 Lausanne, Switzerland e-mail: pedro.romero@licr.unil.ch
J.-P. Rivals · A. Escher · E. Piotet · P. Monnier · L. Bron Service of Head and Neck Surgery,
University Hospital, Lausanne, Switzerland
K. Thielemans
Department of Physiology-Immunology, Medical School of the Vrje Universiteit Brussel, Brussels, Belgium
D. Dojcinovic
Ludwig Institute for Cancer Research Ltd., Lausanne Branch, 1066 Epalinges, Switzerland
of intracellular proteins. One of the most promising catego-ries of TAAs is transcriptionally reactivated genes, not expressed in normal adult host tissues but only present in germline cells and cancer cells and referred as cancer testis antigens (CTA). Their highly restricted tissue expression patterns make them ideal targets for immunotherapy of numerous cancers. Tumor-speciWc vaccines are thus designed to generate T-helper and T-cytotoxic lymphocytes responsive to TAAs. Evidence for the presence of naturally acquired T-cell responses speciWc for these antigens have been demonstrated in patients with various cancers but, to our knowledge, were never studied in HNSCC patients [2–
5]. Generally, the frequency of natural T-cell responses against CT antigens seems to be relatively low in peripheral blood of cancer patients. The natural responses are for this reason diYcult to detect and often require at least one round of in vitro expansion by stimulation with the antigenic pep-tide on appropriate antigen-presenting cells to be detected. Both, the use of overlapping peptide pools and the screening of responding T cells using diVerent cytokines tend to increase the level of natural responses detected in cancer patients that were probably until recently underestimated.
Tumor antigens encoded by the family of MAGE-A genes are of particular interest in cancer immunotherapy, because they are expressed in a variety of malignancies. Among others, these include head and neck squamous cell carcinoma, melanoma or non-small cell lung carcinoma [6,
7]. In a survey performed by our group, tumor biopsies of 52 HNSCC patients showed that MAGE-A3 or -A4 CTA were expressed in 75% of the cases, regardless of the sites of pri-mary tumors such as oral cavity or hypopharynx, conWrming previous results [8]. Since MAGE-A3 is most frequently expressed in major tumor types, as mentioned above, it has been the focus of numerous studies on speciWc T cell-medi-ated immune responses pre- and post-antigen-speciWc immunotherapy [9]. A small proportion of cancer bearing patients naturally develop antibody responses to MAGE-A3, indicating that this tumor antigen is capable of evoking spontaneous immune responses [10, 11]. Moreover, epitope-speciWc memory CD8+ and CD4+ T-cell responses were
rarely detected in cancer patients at relatively low frequen-cies [12–14]. Very few reports to date have documented the precise frequency of MAGE-3 speciWc CD4+ T cells
natu-rally arising in cancer patients [10, 15, 16]. MAGE-A4 is as frequently expressed as MAGE-A3, at least in HNSCC, but has been far less studied. Despite some data indicating an inverse correlation of the expression of MAGE-A4 protein with patient survival [8], no immunotherapeutic protocols have been conducted, mainly because MAGE-A4 speciWc T-cell epitopes have not been described yet.
Naturally acquired immunity against these CTA epitopes in HNSCC patients before vaccination or treatment have not been investigated in detail; yet, the overall prevalence of
these responses would be informative to optimize further peptide vaccination clinical trials. In this regard, based on the MAGE-A3 and -A4 protein tumor expression, a small panel of HNSCC patients recruited before any therapy were screened for spontaneous T-cell responses against MAGE-A3 and MAGE-A4 proteins. Without pre-existing knowl-edge of epitopes and or restriction elements, total patients’ PBMCs were analyzed for MAGE-A3 and -A4 speciWc CD4+ T cells. We could detect speciWc CD4+ T-cell responses against several known as well as undeWned MAGE-A3 and MAGE-A4 epitopes in PBMC from seven out of seven patients analyzed after three rounds of in vitro stimulation with overlapping peptides encoding both pro-teins. Some of the responding T-cell populations were cloned and analyzed for MHC restriction and avidity of antigen rec-ognition. Using speciWc CD4+ T-cell clones, we could also
show that MAGE-A3111–125 and MAGE-A3161–175 epitopes are naturally processed and presented. We identiWed the DP1*04 allele as a new MHC II-restricting element for the MAGE-A3111–125 epitope and we could successfully generate
MAGE-A3111–125/DP1*04 multimer to screen PBMCs and TILs from an HNSCC patient. Altogether, these Wndings indicate that MAGE-A3 and -A4 antigens are promising tar-gets for speciWc immunotherapy of HNSCC.
Materials and methods Cells and tissue culture
Peripheral blood mononuclear cells (PBMCs) were obtained from HNSCC patients (Lausanne University Hospital) after informed consent and from seven healthy donors who served as controls (Blood Transfusion Center, Berne, Swit-zerland). Clinical characteristics of the patients included in this study are described in Table1. None of the patients Table 1 Patient characteristics and tumor antigen expression
¡, negative; +, 1–5%; ++, 5–50%; +++, >50% level of expression
a
Calculated at the surgery date
b
Evaluated by RT–PCR (quantitative assessment was performed as previously described, using 1:10 dilutions of SK-Mel-37 RNA as reference [38])
Patients Agea Gender Tumor localization
Stage MAGE expressionb
A3 A4
LAU 2032 72 M Oral cavity IV + +++ LAU 2042 69 F Oral cavity III ++ +++ LAU 2068 68 F Oral cavity IV +++ +++ LAU 2069 64 F Oropharynx III +++ ++ LAU 2073 54 M Oropharynx IV +++ ++
LAU 2086 68 M Larynx III ¡ +++
received immunotherapy before sampling of blood. HNSCC autologous tumor cell lines were generated from surgically removed primary tumor biopsies. Epstein–Barr virus-trans-formed lymphoblastoid B-cell lines (LCLs) were established in our laboratory. LCLs and tumor cells were retrovirally transduced with a pMFG vector encoding huIi80MAGE-3-Ires-tNGFR [17]. Tumor and LCLs cells were maintained in continuous in vitro culture in RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 10% heat-inactivated FCS (GIBCO, Invitrogen) L-glutamine
(2 mM), penicillin (100 U/ml) and streptomycin (50g/ml; Biowhittaker, Walkersville, MD, USA).
HLA typing
HLA class II typing was performed at Geneva’s University Hospital by J. M. Tiercy, on genomic DNA extracted from frozen PBLs isolated from patient’s blood samples. HLA-DR1 and -DQ1 low-resolution typing (two-digit) was performed by reverse PCR sequence-speciWc oligonucleo-tide hybridization using Luminex technology (OneLambda; Ingen). High-resolution typing (four digit) for HLA-DR1, -DP1 and -DQ1 loci was achieved by PCR sequence-speciWc primers using Genovision reagents (Milan Analytica). Synthesis of MAGE-A3 and MAGE-A4 peptides
and peptide pools
MAGE-A3 and MAGE-A4 15 mers peptides overlapping by 10 amino acids were synthesized by Proimmune (UK). Selected sequences were synthesized by the stepwise solid phase method and synthetic peptides were puriWed by semi-preparative reverse-phase HPLC. All peptides were >90% pure as indicated by analytic HPLC. The peptides were lyophilized, then reconstituted in DMSO at 10 mg/ml or aliquots of 1 mg/ml and stored at ¡20°C. To reduce the number of individual peptide stimulation, we generated six peptide pools with ten peptides each for MAGE-A3 or MAGE-A4 overlapping peptides.
In vitro propagation of speciWc CD4+ T cells
Twelve pools of 10 MAGE-A3 or MAGE-A4 synthetic pep-tides were used to stimulate the PBMCs from the HNSCC patients and HDs. Twenty million PBMCs, depleted for CD25+ T cells using anti-CD25 microbeads and MiniMACS magnetic separation columns (Miltenyi Biotec), were cul-tured for 14 days in RPMI 1640 (Gibco, Grand Island, NY, USA), supplemented with heat-inactivated pooled human serum (8%), L-glutamine (2 mM), penicillin (100 U/ml) and
streptomycin (50g/ml; Biowhittaker, Walkersville, MD, USA), containing MAGE-A3 or MAGE-A4 peptide pools (2M of each peptide). On day two, half medium volume
from each well was removed and replenished with fresh tis-sue culture medium (TCM) containing interleukin (IL)-2 (150 UI/ml) (Chiron), without any further antigen stimula-tion. Cultures were expanded for 14 days and restimulated two more times with the same amount of MAGE-A3 or MAGE-A4 peptide pools pulsed on irradiated (3,000 rad) autologous PBMCs as antigen-presenting cells (APCs). IFN-/TNF- intracellular cytokine staining
On days 7–10 after the third round of simulation, cultures were tested for the presence of speciWc CD4+ T cells with
the same pool of peptides (2M each) in an IFN- and TNF- intracellular cytokine staining assay performed as follows. Cells were stimulated for 18 h at 37°C in the pres-ence or abspres-ence of the peptide pools and 10g/ml brefeldin A (Sigma–Aldrich) was added after the Wrst hour of incuba-tion. After incubation and washing, cells were stained with anti-CD4-PE-Cy7 and anti-CD3-APC-Cy7 mAb (Beckman Dickinson) for 20 min at 4°C, and then Wxed with 1% form-aldehyde, 2% BSA and 5 mM azide in PBS. Cells were then stained intracellularly with anti-IFN--PE and anti-TNF--APC mAb (Beckton Dickinson) in the presence of 0.1% saponin (Sigma–Aldrich). Samples were analyzed by Xow cytometry on a Facs Array and data were analyzed using Flow Jo software (Beckton Dickinson).
Generation of clones and antigen recognition assay CD4+ T cells producing IFN- in the presence of the pool of peptides were cloned using IFN- and TNF- secretion assays (Miltenyi Biotec) after 6 h of peptide stimulation by Xow cytometry-based sorting using a FACSVantage SE (Becton–Dickinson, Sunnyvale, CA, USA). Sorted cells were cloned by limiting dilution and growing clones were screened for IFN- and TFN- release in the pres-ence or abspres-ence of indicated MAGE-A3 or MAGE-A4 peptides.
For each functional assay, 5,000–20,000 of growing MAGE-A3 or MAGE-A4 speciWc CD4+ T-cell clones were
used. For recognition of indicated target cells, equal num-bers of antigen-presenting cells or tumor cell lines were pulsed or not with indicated peptides, extensively washed and then added to the T cells. In peptide titration experi-ments, the following concentrations of peptide were added: 50–25–13–6–1.5 and 0.7M. Where indicated, superna-tants of anti-HLA-DR (D1.12), -DP (B7.12.1) or -DQ (BT3.4) were used to speciWcally block MHC class II rec-ognition. IFN- release was assessed in the culture superna-tant after 24 h of incubation at 37°C by ELISA (hu IFN- Cytoset kit; BioSource Europe) following the manufac-turer’s instructions and using Xat-bottom 96-well Nunc-Immuno plates (Apogent).
Generation of HLA-DP1*04 multimers and multimer labeling
The generation of HLA-DP1*04 multimers was per-formed as already described [18] using the MAGE-A3111–125 peptide. Multimer labeling was performed for 1 h at 37°C before staining with Xuorescent mAbs directed against surface molecules (anti-CD4-FITC) (20 min at 4°C). PI labeling was used to exclude dead cells. Cells were analyzed by Xow cytometry on a FAC-Scan (BD Biosciences) using Cell Quest software (BD Biosciences).
Results
MAGE-A3 and MAGE-A4 speciWc CD4+ T-cell responses
in head and neck cancer patients
To assess the naturally acquired anti-tumor T-cell responses taking place in head and neck squamous cell carcinomas patients (HNSCC), tumor biopsies were Wrst screened for tumor-associated antigen expression by RT–PCR and immunohistochemistry to identify putative immunogenic antigens. MAGE-A3 and -A4 tumor anti-gens were speciWcally expressed in about 70% of the patients (MAGE-A3 in 51%, MAGE-A4 in 60% [8]). We assessed T-cell responses speciWc for these two well-represented antigens in peripheral blood from seven HNSCC patients, selected upon their MAGE-A3 and/or -A4 high tumor expression (Table1). BrieXy, CD25+
cell-depleted PBMCs from these patients were stimu-lated with overlapping peptides covering the entire sequence of the two proteins. These peptides were 15 amino acids long, overlapping by 10 amino acids and grouped into six pools containing 10–11 peptides. CD25+ T-cell depletion was performed prior to stimula-tion since these cells have been reported to limit the in vitro expansion of antigen-speciWc CD4+ T cells [19]. A
side-by-side comparison of CD25+ replete and depleted
PBMCs samples was not possible due to the limited amount of blood allowed by the clinical protocol. After three rounds of stimulation, the repertoire of epitopes recognized by polyclonal CD4+ T cells was determined by testing their reactivity to each pool and then each peptide forming the pool in a 6-h IFN-/TNF- intracel-lular cytokine staining (ICS) assay using a Wve-color immunoXuorescence labeling protocol. The frequency of CD4+ T cells producing IFN- or TNF- in the
responding patients ranged from 2 to 64% after the third stimulation. In contrast, undetectable or very low fre-quency of IFN- or TNF- positive cells was found in healthy controls and in patients’ PBMCs stimulated with
irrelevant or no peptides (range 0–2%). An example of the data obtained following this analysis for one patient and one healthy donor are shown in Fig.1a.
Among the seven HNSCC patients tested, all of them showed detectable CD4+ T-cell responses against at least one MAGE-A3 or MAGE-A4 peptide. Four patients, LAU 2032, LAU 2042, LAU 2073 and LAU 2091, showed responses against both MAGE-A3 and MAGE-A4 pep-tides. Responses against MAGE-A3281–295 peptide and MAGE-A4301–315 peptide appeared simultaneously in two diVerent patients despite their diVerent class II MHC alle-les, suggesting recognition of promiscuous epitopes. Most of the detected MAGE-A3 responses were speciWc to previ-ously found and/or described epitopes, such as 111–125 and 116–130 [17, 20], region 141–155 [20], 146–160 [20,
21], 161–175 [20, 22] and 281–295 [20, 21]. In contrast, three responses were speciWc to new epitopes spanning the regions 156–170, 216–230 and 241–255. All of the detected MAGE-A4 responses were speciWc to new epi-topes covering a large part of the protein, from region 116 to 317 (Fig.1b).
To further characterize these epitopes, clones were derived from the primary cultures that were able to respond to shortterm stimulation with the selected MAGEA3 and -A4 peptides using the IFN-/TNF- capture assay and Xow cytometry-assisted cell sorting.
CD4+ T-cell responses against MAGE-A3 and -A4 peptides
are mainly HLA-DR and -DQ restricted
After cloning, ten growing clones that recognized dis-tinct MAGE-A3 peptides (111–125, 146–160, 161–175, 216–230, 281–295) and MAGE-A4 peptides (271–285, 286–300, 291–305, 301–315, 303–317) could be main-tained in culture and characterized. To identify the HLA restricting allele of the diVerent peptide epitopes, grow-ing CD4+ T-cell clones were tested in short-term stimu-lation with the selected MAGE-A3 or -A4 peptides in the presence of either anti-HLA-DR-, -DP- or -DQ-spe-ciWc Abs known to speciWcally block antigen recogni-tion. The MAGE-A4291–305 speciWc CD4+ T-cell clone from LAU 2069 recognized the peptide in an HLA-DR restricted manner (Fig.2a). In addition, to identify the restricting allele, we challenged CD4+ T-cell clones with a panel of LCLs with identiWed class II MHC, pulsed with individual peptides. Using diVerent combinations of presenting LCLs, we could determine that the clone from LAU 2069 recognized the MAGE-A4291–305 peptide presented by HLA-DR1*13 restricted allele (Fig.2b).
Clones from LAU 2091 recognized the MAGE-A3146–160 peptide in an HLA-DR1*07 restricted manner, while they recognized the MAGE-A4271–285, MAGE-A4301–315 and
MAGE-A4305–317 peptides presented by HLA-DQ1*02. In
addition, MAGE-A3111–125 peptide, described as an HLA-DR1*13 epitope by Chaux et al. [17], appeared to also be present by the HLA-DP1*04 allele. The clone from LAU 2073 recognized the MAGE-A3281–295 peptide presented by
HLA-DR1*08 and the clone from LAU 2032 recognized the same peptide presented by HLA-DR1*04. The MAGE-A3216–230 and MAGE-A4286–300 peptides, recog-nized by CD4+ T-cell clones from LAU 2032, are presented
by HLA-DR1* 04. Finally, clones from LAU 2068 and LAU 2069 recognized the MAGE-A3161–175 and MAGE-A4291–305 peptides presented by HLA-DR1*07 and HLA-DR1*13, respectively (Fig.2c).
Characterization of MAGE-A3 and MAGE-A4 speciWc CD4+ T-cell clones
To more precisely deWne the epitopes recognized by the clones from head and neck cancer patients, we stimulated the MAGE-A3 and MAGE-A4 speciWc CD4+ T-cell clones with graded amount of peptides followed by assessment of IFN- production in the culture supernatants. Peptide titra-tion curves of MAGE-A3146–160 and MAGE-A4301–315 spe-ciWc CD4+ T-cell clones from LAU 2091 are shown in
Fig.3a. Both clones recognized their corresponding syn-thetic peptides with relatively low avidity in the micromo-lar range (EC50> 10M and EC50> 7M, respectively).
Fig. 1 Pre-existing CD4+ T-cell responses against MAGE-A3 and
MAGE-A4 antigens in HNSCC patients. a PBLs from HNSCC pa-tients were stimulated in vitro with MAGE-A3 and MAGE-A4 over-lapping peptide pools. After three rounds of stimulation, cultures were tested by intracellular cytokine staining for IFN- and TNF- secretion in response to the presence of peptide pools. Cytokine secretion of
CD4+ CD3+ lymphocyte gated populations from one HNSCC patient
and one healthy donor in response to peptide pool and individual pep-tide are shown as example. b Summary of the responses detected after in vitro stimulation in seven analyzed HNSCC patients. The responses that could be further cloned by cell sorting and limited dilutions are indicated as black boxes. Undescribed epitopes are depicted as “NEW”
MAGE-A3146–160 speciWc CD4+ T-cell clone failed to sig-niWcantly recognize the neighboring overlapping peptides and the corresponding peptide in the MAGE-A4 protein. The MAGE-A4301–315 speciWc CD4+ T-cell clone
recog-nized also the 303–317 region and failed to signiWcantly recognize the neighboring overlapping peptides and the corresponding peptide region in the MAGE-A3 protein suggesting that the minimal epitope for this clone was located between the amino acids 303 and 315. The analysis of the other MAGE-A3 and MAGE-A4 speciWc CD4+
T-cell clones showed that those clones recognized their respective peptides with low avidity in a micromolar range (Fig.3b).
Both MAGE-A3111–125 and MAGE-A3161–175 epitopes are naturally processed and presented by the MHC class II pathway
We wanted to determine whether the identiWed epitopes were naturally processed and presented by MHC class Fig. 2 MHC class II restriction
of MAGE-A3 and -A4 speciWc CD4+ T clones. a Example of
class II restricted allele assess-ment on LAU 2069 CD4+ T-cell
clone 1 speciWc for MAGE-A4291–305. Where indicated,
anti-DP, DQ, DR puriWed antibodies were pre-incubated with cells prior to peptide stimulation. IFN- release was measured in the supernatant by ELISA. b Example of Wne HLA II restriction performed on LAU 2069 clone 1. HLA class II matched LCL cell lines were pulsed (black bars) or not (white bars) with MAGE-A4291–305
peptide and presented to the speciWc clone. Recognition of the LCL cells with the restricting allele was measured by ELISA IFN-. c Summary table of MHC class II restriction of the cloned responses
II-positive cells. Autologous monocyte-derived DC, gener-ated after CD14+ cell selection and maturation were used as APC in peptide recognition assays and pre-incubated with the MAGE-A3 recombinant protein or with an irrelevant Flu recombinant protein used as control. As illustrated in Fig.4a, monocyte-derived DC were able to eYciently
pres-ent the recombinant MAGE-A3 protein to the CD4+ T-cell clone from LAU 2068 speciWc for MAGE-A3161–175 pep-tide on HLA-DR dependent manner, indicating that this epitope was naturally processed and presented. Unfortu-nately, MAGE-A4 epitopes could not be studied with this method since no MAGE-A4 recombinant protein was avail-able.
The natural processing of diVerent epitopes could also be assessed using HLA-matched LCLs engineered to express either MAGE-A3 or MAGE-A4 in the endosomal–lyso-somal compartment [17]. The MAGE-A3111–125 speciWc CD4+ T-cell clone from LAU 2091 recognized the peptide in DP1*04 LCLs transduced with MAGE-A3, conWrming that the MAGE-A3111–125 peptide was processed through
the class II presentation pathway (Fig.4b). In addition, MAGE-A3146–160, MAGE-A3216–230 and MAGE-A3281–295 epitopes were not processed (Fig.4c). The MAGE-A4286–300
peptide identiWed on CD4+ T cells from LAU 2032 was not processed by LCLs engineered to express MAGE-A4. Finally, the processing of MAGE-A4271–285, 301–315 and 303–317 peptides identiWed on CD4+ T cells from LAU 2091 could
not be addressed, since no HLA-DQ LCLs engineered to express MAGE-A4 were generated. MAGE-A4291–305 epi-tope could not be addressed since the speciWc clone could not be maintained in culture (Fig.4c).
Memory CD4+ T cells speciWc for MAGE-A3111–125/DP4 are present after in vitro stimulation of PBMCs
of a HNSCC patient
The MAGE-A3111–125 peptide was used to prepare HLA-DP1*04 multimer, which was tested on clones gener-ated from LAU 2091 and additional nonspeciWc clones or negative controls. Several independent CD4+ T-cell Fig. 3 Peptide titration and
cross reactivity of MAGE-A3 and -A4 speciWc CD4+ T-cell
clones. a Peptide titration exper-iments were performed using clone-speciWc MAGE-A3 sequence (open symbol) and compared to both neighboring overlapping sequences and the MAGE-A4 or MAGE-A3 homologous sequence. Shown are IFN- titration curves expressed as percentage maximal IFN- release of two representative clones generated from peripheral blood of patient LAU 2091. b Summary of the EC50 of the diVerent clones
clones speciWc for MAGE-A3111–125 peptide were stained
by the multimer and no staining was observed on nega-tive clone 30 (Fig.5a). In addition, no staining was observed on irrelevant DP4/tetanus toxoid-speciWc clone 4 (Fig.5b). We next analyzed ex vivo with the
MAGE-A3111–125 multimer, PBMCs from DP1*04 LAU 2091
patient in which we detected speciWc CD4+ T cells after
in vitro stimulation (Fig.5c, left panel). No ex vivo detectable responses against DP4/MAGE-A3111–125 could be detected in PBMCs and TILs of this patient nor in the other head and neck cancer patients (data not shown). Finally, combining the multimer staining with pheno-typic marker analysis, we analyzed the three in vitro PBMCs stimulation from LAU 2091 used to generate the speciWc clones against MAGE-A3111–125 peptide. The
speciWc CD4+ T cells, which were initially detected after three rounds of in vitro stimulation by cytokine secretion
assays, could already be detected after the Wrst round of in vitro stimulation by using the HLA-DP1*04/A3111–125 multimer. The speciWc population increased by approxi-mately fourfold after each stimulation with the peptide pools from 0.8 to 11%. We found that all MAGE-A3111–125/DP1*04 tetramer-positive cells had a memory phenotype, as they were all CD45RA¡. In addition, the
CCR7+ expression was downregulated upon antigenic stimulation concomitant with the diVerentiation of cen-tral memory to eVector memory cells (Fig.5c, right panel).
Discussion
The main Wnding in this study is the demonstration of fre-quent naturally acquired T-cell immunity in advanced Fig. 4 Natural processing and
presentation of identiWed MAGE-A3 and -A4 peptides. a Mature HLA-matched den-dritic cells were activated with LPS and pulsed with 5M of recombinant MAGE-A3 protein. After washing, DCs were cocultured with CD4+
T-cell clones at 1:1 ratio. IFN- production was measured after 20 h in the supernatant by ELISA. Shown are example of MAGE-A3 161–175 peptide processing by HLA-DRB1*07 mature DC and presentation to LAU 2068 clone 4.
b Recognition of the endoge-nous MAGE-A3 antigen was assessed upon HLA-matched EBV cells transduction with a retroviral construct encoding Ii.MAGE-A3 and coculture with MAGE-A3 peptide-speciWc clones. Shown is example of LAU 2091 clone 1 speciWc for epitope 111–125 cocultured with HLA-matched RULE LCL transduced with MAGE-A3. c Summary table of the described epitopes and natural processing
HNSCC patients to the two most frequently expressed shared tumor-speciWc antigens in HNSCC, namely MAGE-A3 and -A4. Indeed, speciWc CD4+ T cells could be readily
expanded from PBLs from all seven HNSCC chosen based on the expression of the MAGE genes by their tumors. This was not the case when PBLs from a group of healthy donors were subjected to the same in vitro stimulation pro-tocol. Together, our data suggest the existence of memory responses to MAGE-derived antigens. In support of this notion, monitoring of MAGE-speciWc T cells using Xuores-cent multimers during the consecutive in vitro stimulations showed the presence of expanded T cells already 10 days after the Wrst round of stimulation with peptides. As is the case for numerous epitopes derived from tumor antigens, the frequency of memory T cells is below the detection
limit by multimers-guided Xow cytometry, but can be readily revealed by in vitro expansion of speciWc cells driven by the antigen. Exact determination of the actual frequency of MAGE-speciWc T cells in these patients would require either limiting dilution analysis coupled to multimer label-ing, or enrichment of rare multimer events from a large number of PBLs, as reported in mouse models [23].
While there are several reports identifying multiple epi-topes recognized by MHC class I restricted MAGE-A4 spe-ciWc cytotoxic T lymphocytes [24–26], only one paper identiWed helper epitopes in a peptide derived from the MAGE-A4 tumor antigen [27]. In the present study, up to 13 distinct class II MAGE-A4 epitopes were recognized by CD4+ T cells in six to seven patients analyzed. With the exception of the 281–295 peptide described by Ohkuri Fig. 5 Detection of MAGE-A3111–125 speciWc CD4
+
T-cell clones with MAGE-A3 111–125/DPB1*04 multimer. a SpeciWc (clones 131, 134, 48) and nonspeciWc (clone 30) clones from LAU 2091 generated against MAGE-A3111–125 were labeled with DP4/MAGE-A3111–125 -PE
multimer and anti-CD4-FITC antibodies. Cells were gated on live CD3+ cells. b Fine speciWcity of DP4 tetramer shown by distinct stain-ing of tetanus toxoid947–960 and MAGE-A3111–125 CD4
+
T-cell clones (clone 4 and 13, respectively) with a combination of DP4/TT947–960-APC
and DP4/MAGE-A3111–125-PE multimers. Cells were gated on live
CD4+ T cells. c DP4/MAGE-A3111–125 speciWc CD4 +
T cells detected after 1, 2 and 3 rounds of in vitro stimulation of the LAU 2091 PBLs with MAGE-A3 overlapping peptide pools. Left dot plots PBLs were screened with the tetramer and gated on live CD4+ T cells. Right dot plots phenotype of CD4+ tetramer T-cell populations analyzed by CD45RA and CCR7 staining. All dot plots are gated on live cells
et al., all are undescribed MAGE-A4 derived T-cell epitopes. We obtained clonal CD4+ T-cell populations speciWc for Wve epitopes (MAGE-A4: 271–285, 286–300, 291–305, 301–315 and 303–317) presented by HLA class II DQ1*02, DR1*04, DR1*13, DQ1*02 and DQ1*02, respectively. Some of these peptides may, however, be pre-sented by more than one MHC class II molecule. For instance, speciWc CD4+ T cells against the MAGE-A4
301–315
epitope were observed in two cancer patients. In one patient, LAU 2091, the epitope was presented by HLA-DQ1*02. However, the second patient, LAU 2073, was HLA-DQ1*02 negative, thus implying that speciWc T-cell recognition involves a diVerent MHC class II allele.
Only one HLA-matched LCL engineered to express MAGE-A4 in the endosomal–lysosomal compartment could be tested for natural processing and presentation of MAGE-A4 epitopes. However, the MAGE-A4286–300 spe-ciWc CD4+ T-cell clone did not detectably recognize this
MAGE-A4 expressing LCL. These results might suggest that the peptide is not eYciently processed in the endo-somal compartment of transduced B cells. In this regard, the other report on MAGE-4 helper-speciWc T cells used autologous monocytes-derived dendritic cells pulsed with the recombinant MAGE-A4 protein to determine speciWc MAGE-A4 CD4+ T cells and used overlapping peptides to identify the minimal MAGE-A4284–293 epitope [27]. This Wnding indicates that peptides in this region of the protein can be processed for MHC class II loading and presentation to T cells. Thus, an alternative possibility is that the CD4+ T-cell clones obtained from cancer patients used in this study are of too low avidity to pick up processed peptides in MAGE-A4 transduced B cells.
In contrast to MAGE-A4 epitopes, MAGE-A3 epitopes have been extensively studied in terms of speciWc T-cell responses. Indeed, several class I and class II epitopes have been described both in healthy donor and in cancer patients. In this study, we could detect CD4+ T-cell responses against six previously described MAGE-A3 epitopes (111– 125, 116–130, 141–155, 146–160, 161–175 and 281–295) as well as three new epitopes (MAGE-A3: 156–170, 216– 230 and 241–255). The MAGE-A3111–125 and MAGE
A3161–175 peptides appeared to be immunodominant
epitopes. Indeed, these were shown to be naturally processed by diVerent types of APCs and presented by HLA-DP1*04 and HLA-DR1*07, respectively. The MAGE-A3111–125 epitope has been identiWed Wrst as an epi-tope presented by HLA-DR13 [17], then also by HLA-DR1, HLA-DR4, HLA-DR11 and HLA-DR7 molecules [20]. The MAGE-A3111–125 peptide is therefore another example of a promiscuous CD4+ T-cell epitope. Our data show that in addition to presentation by multiple HLA-DR allelic prod-ucts, this peptide can also be presented by HLA-DP4. The HLA-DP1*04 allele is expressed by approximately 60%
of the Caucasian population and covers, together with the frequent HLA-DR1*01 (20%), HLA-DR1*04 (27%), HLA-DR1*07 (27%), DR1*11 (19%) and HLA-DR1*13 (21%) alleles, a signiWcant fraction of Caucasian patients. Moreover, the MAGE-A3 sequence 111–125 con-tains also the HLA-A2 (MAGE-A3112–120) binding epitope [28]. We demonstrated here the ability of Xuorescent DP4/
MAGE-A3111–125 peptide multimers to identify speciWc
CD4+ T cells propagated in vitro, thus opening the way to monitoring of these responses.
Concerning the MAGE-A3161–175 sequence, several pre-dicted MHC class II binding epitopes have been described [22]. It also contains HLA-A1 (MAGE-A3168–176) and HLA-B44 (MAGE-A3167–175) restricted epitopes [29], sug-gesting an interesting peptide for vaccination. However, natural processing of the class II peptide could not be dem-onstrated in one study [20]. Another group reported that it was poorly formed and did not Wnd evidence of speciWc CD4+ T-cell responses in advanced melanoma patients [22]. Our results are in sharp contrast with these previous reports since we could detect speciWc CD4+ T cells and
could also show recognition of processed recombinant MAGE-A3 protein. These discrepancies may be due to the diVerent types of cancer.
In contrast to the former epitopes, the MAGE-A3146–160 speciWc, MAGE-A3216–230 speciWc and MAGE-A3281–295
speciWc CD4+ T cells failed to recognize in vitro the
MAGE-A3 protein after processing by autologous dendritic cells. However, reports from other groups showed eYcient processing of the MAGE-A3146–160 and MAGE-A3281–295 epitopes [20, 21, 30]. Again, it might be that the CD4 T-cell clones recovered from this series of head and neck cancer patients bear TCRs of relatively low avidity.
Despite recognition of processed peptides in recombi-nant protein fed, or MAGE-A3 transduced antigen-present-ing cells, no tumor recognition could be demonstrated in the few CD4+ T-cell clone/tumor combinations that could be tested (Table2 and data not shown). This could not be explained by lack of expression of MHC class II molecules, as expression could be detected at the cell surface and their levels could be increased upon IFN- treatment. These results suggest that priming of the memory responses detected in cancer patients probably required processing and presentation by professional APCs from internalized apoptotic tumor cells. They also imply indirect antigen rec-ognition by eVector CD4+ T cells at the tumor sites.
How-ever, it cannot be excluded that high avidity CD4+ T cells against at least some of the MAGE-derived epitopes can directly recognize MHC class II tumor cells. This possibil-ity is reinforced by reports from other groups who have succeeded in showing direct tumor antigen recognition by MAGE antigen-speciWc CD4+ T cells. Concerning the
presence of detectable speciWc CD4 T-cell responses, all but one responding patients expressed the target gene prod-uct (Table1). The single exception may simply reXect the
incomplete covering of antigen expression by the small biopsy that may not be representative of the entire tumor mass.
Epitope-speciWc CD8 T-cell responses were rarely detected from freshly isolated peripheral blood in cancer patients and was found to be quite low before vaccination [12, 14, 31]. However, only after in vitro culture or 3 weeks post-vaccination, CD8+ T cell could be expanded and their functional status estimated [10, 32]. The apparent paucity of MAGE-speciWc CD8+ T-cell responses in this series
of HNSCC patients might suggest the existence of CD8+ T-cell tolerance to these antigens. However, we favor the possibility that this paucity only reXects the inadequacy of peptide pools made of 15-mer peptides to eYciently expand not only CD4+ T cells, but also MHC class I restricted CD8+ T cells. To address this issue, we are planning to
revisit MAGE-speciWc CD8+ T-cell responses in these patients using pools of nine and ten amino acid long syn-thetic peptides covering the MAGE-A3 and -A4 proteins.
In all, very few reports to date have documented the pre-cise frequency of MAGE-A3 speciWc CD4+ T cells natu-rally arising in cancer patients and none for MAGE-A4. By studying a large panel of cytokine produced by peripheral PBLs in responses to tumor-associated antigens, Inokuma et al. [15] were able to detect ex vivo signiWcant CD8 and CD4 T-cell responses against MAGE-A3 in 17/23 breast cancer patients naïve to immunotherapy. Similarly, another study reported the baseline level of CD4+ T-cell responses
prior MAGE-A3 protein and adjuvant immunization of melanoma patients and could detect MAGE-A3 speciWc helper T-cell responses in 3/16 patient before vaccination [16]. In contrast, Atanackovic et al. [10] found only 1 out of 18 non-small cell lung cancer patients vaccinated with
MAGE-A3 protein and adjuvant, having a pre-existing anti-body response against MAGE-A3 protein as well as CD4 T-cell responses to MAGE-A3 DP4 epitope (p243–258). Globally, spontaneous and detectable anti-MAGE-A3 responses seem to be a rare event in cancer patients; how-ever, some patients may have mounted a response below the detection level of the techniques used at the time of analysis. In our experiments, the requirement of three con-secutive rounds of in vitro stimulation to detect MAGE-A3/ A4 speciWc CD4+ T cells reXect a relatively low frequency
of these cells as conWrmed by the ex vivo absence of detect-able DP4/MAGE-A3111–125 peptide multimer+ CD4+ T
cells. However, it is also likely that these CD4+ T cells were anergic in vivo and reacquired full functional competence upon repeated in vitro stimulation with speciWc peptide and IL-2. In agreement with this notion, HNSCC have indeed been shown to be highly immunosuppressive tumors. Among the many immunosuppressive mechanisms, the impairment of T-cell activation and the production of immune inhibitory mediators by the tumors have been fre-quently observed in HNSCC [33–35] and would Wt with the
generation of low avidity of CD4+ T cells as detected in this study.
Vaccination with class II MAGE-A3 speciWc peptide or recombinant MAGE-A3 proteins have shown no signiWcant toxicity and has led to the induction of strong tumor-spe-ciWc CD4+ T-cell responses [10, 16, 36, 37]. Thus, targeting
MAGE-A3 and/or -A4 for immunotherapy is an attractive possibility in patients with HNSSC tumors. Sequences such as MAGE-A3111–125 and MAGE-A3161–175 bear naturally processed epitopes that are recognized by both MHC class II restricted CD4+ T cells and MHC class I restricted CD8+ T cells. Approaches to antigen delivery such as recombi-nant protein or long synthetic peptides may provide potent immunogens able to induce broad T-cell responses in a large segment of the patient population, thus alleviating the constraints imposed by MHC restriction. These results also provide a useful baseline for future immunomonitoring studies in vaccinated patients.
Acknowledgments We thank Ms. Eliane Cottin for her precious technical assistance and Dr. Donata Rimoldi for assessment of TAA expression in tumor cells and her valuable suggestions on tumor cell explants and culture.
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